1. Field of the Invention
The present invention relates to immunomodulation and to the stimulation and enhancement of the immune or inflammatory response, including the use of adjuvants to enhance immune response to a vaccine. The present invention also relates to treatment of injuries, diseases, disorders and conditions that result in neurodegeneration.
2. Description of the Related Art
Millions worldwide are affected with infectious diseases, cancer, lymphomas, HIV, AIDS, rheumatoid arthritis, asthma, immunodeficiency disorders and diseases involving defective immune, allergic, or inflammatory responses. Many diseases and their disease outcomes involve immune or inflammatory responses and are associated with the stimulation of dendritic cells (DCs), T cells, the production or suppression of various cytokines, chemokines and interferons, and the increase or decrease in the availability of cytokines and chemokine receptors. In addition, many neurological and neurodegenerative diseases involve damage to nerve or neuronal cells.
Dendritic Cells
Dendritic cells (DCs) are the most potent antigen-presenting cells and they play a crucial role in the generation and regulation of immunity (Banchereau and Steinman, 1998; Sallusto and Lanzavecchia, 1994). Their priming ability is acquired upon maturation and is characterized by the activation of transcription factors, antigen processing, control of migration and regulation of inflammatory responses (Shutt et al., 2000; Granucci et al., 2001; Sallusto et al., 1999; Ouaaz et al., 2002). Regulated migration of DCs is central to the induction of physiological immune responses. The expression of surface molecules on DCs known to be critical for antigen-presenting function include HLA-DR, CD40, CD83, CXCR4 and CD80 and CD86 and this is associated with increased cytokine and chemokine production and stimulatory capacity.
DCs link innate and adaptive immunity by sensing pathogens or vaccinogens and signaling a variety of defense responses. DCs comprise a family of cells specializing in antigen capture and presentation to T cells, play a role in bacterial uptake across mucosal surfaces, can open tight junctions and sample antigens directly across epithelia (Rimoldi et al., 2004). DCs sample enteric antigens in the lamina propria and Peyer's patches, and transport them to mesenteric nodes where they are presented to lymphocytes (Macpherson et al., 2004). DCs are potent antigen-presenting cell that are able to initiate and modulate immune responses and are hence often exploited as cellular vaccine components for applications such as immunotherapy. Their ability to migrate from peripheral tissues to the T cell areas of draining lymph nodes is crucial for the priming of T lymphocytes. Signal molecules that promote DCs to acquire potent Th-1 cell stimulatory activity and substantial chemotactic responsiveness to chemokines would be useful in the development of vaccines and for tumor immunotherapy (Scandella et al., 2002).
DCs are the first target of HIV and, by clustering and activating T cells, may both activate antiviral immunity and facilitate virus dissemination (Sewell and Price, 2001; Frank and Pope, 2002). During HIV infection, there is loss of immune control and dysfunction of DCs may contribute to immune suppression associated with AIDS progression (Quaranta et al., 2004). Activation of immature DCs by manipulating their phenotypical, morphological and functional developmental program would have useful clinical applications for therapeutic intervention for AIDS patients.
Cytokines and Costimulatory Molecules
Cytokines are proteins that regulate immune and inflammatory reactions. Cytokines play an essential role in the activation and maintenance of both innate and acquired immune responses. Cytokines and chemokines have been used as vaccine adjuvants with both traditional and DNA vaccines. Cytokines are small proteins (˜25 kDa) that are released by various cells in the body, usually in response to an activating stimulus, and induce responses through binding to specific receptors. They can act in an autocrine manner, affecting the behavior of the cell that releases the cytokine, or in a paracrine manner, affecting the behavior of adjacent cells. Some cytokines can act in an endocrine manner, affecting the behavior of distant cells, although this depends on their ability to enter the circulation and on their half-life.
Interleukin-12 (IL-12) is a potent enhancer of cellular responses. IL-12 is a potent proinflammatory cytokine with potent antitumor effects that enhances cytotoxic T lymphocytes (CTL) and natural killer (NK) cell activity. IL-12 treatment of mice augments antibody responses to T independent polysaccharide antigen (Buchanan et al., 1998). IL-12 and IL-1 have been shown to induce systemic immunity to mucosally administered vaccines (Boyaka and McGhee, 2001). Studies have shown the regression of established neuroblastoma in mice vaccinated with IL-12 transduced dendritic cells (Redlinger et al., 2003). Another study with syngeneic A/J mice using intratumorally injected IL-12 transduced cells showed that mice underwent tumor regression indicating that increased IL-12 production by DCs induces a significant antitumor response in a poorly immunogenic murine model of neuroblastoma (Shimizu et al., 2001). These results clearly show the vital role of DCs in the immunobiology of neuroblastoma, and that protection of these cells from tumour induced apoptosis is a critical aspect for immunotherapies treating aggressive tumors. Co-expression of cytokines, chemokines and costimulatory molecules enhances the immunogenicity of DNA vaccines.
As is true for most intracellular pathogens, immunization with live Chlamydia trachomatis induces a stronger protective immunity than immunization with inactivated organism and is associated with high levels of the proinflammatory cytokine IL-12 and the enrichment of DCs among mice immunized with viable organisms (Zhang, et al., 1999). These results indicate that the induction of proinflammatory cytokines and activation and differentiation of DCs is important for inducing active immunity to C. trachomatis infection.
Chemokines are a class of cytokines that have chemoattractant properties, inducing cells with the appropriate receptors to migrate toward the source of the chemokine. Certain chemokines may recruit cells to sites of infection. Chemokines such as RANTES may promote the infiltration into tissues of a range of leukocytes including effector T cells. Effector T cells that recognize pathogen antigens in the tissues produce cytokines such as TNF-α, which activates endothelial cells to express E-selectin, VCAM-1, and ICAM-1, and chemokines such as RANTES, which can then act on effector T cells to activate their adhesion molecules.
Chemokines exert their effects through at least nineteen G protein-coupled receptors (GPCRs). The nomenclature of the chemokine receptors follows the notation used for the chemokine subfamilies and they are termed CCR1-10 (CC chemokine receptor 1-10), CXCR1-6, XCR1 and CX3CR1. A remarkable feature of the chemokine receptors is their relative lack of selectivity in ligand binding, with many chemokine receptors binding more than one chemokine with high affinity. For example, eleven chemokines are reported to bind to the CCR1 receptor, including MIP-1α (macrophage inflammatory protein 1α), MIP-1β, MIP-1δ, RANTES (regulated on activation normal T cell expressed and secreted), MCP-1 (monocyte chemotactic peptide 1), MCP-2, MCP-3, MCP-4, Lkn-1 (leukotactin-1), MPIF-1 (myeloid progenitor inhibitory factor 1) and HCC-1 (hemofiltrate CC chemokine 1), with varying affinities and acting with different degrees of agonism. Similarly, individual chemokines act as ligands for different receptors. For example, MCP-3 acts as a ligand for CCR1, CCR2, CCR3 and CCR5. This promiscuity and the apparent redundancy of signaling that may arise poses many questions as to the control of chemokine signaling in different tissues expressing different combinations of chemokines, receptors and effectors (ACTA BIOCHIMICA et BIOPHYSICA SINICA 2003, 35(9):779-788).
There are different variants of HIV, and the cell types that they infect are determined to a large degree by which chemokine receptor they bind as co-receptor. The variants of HIV that are associated with primary infections use CCR5, which binds the CC chemokines RANTES, MIP-1α, and MIP-1β, as a co-receptor, and require only a low level of CD4 on the cells they infect. These variants of HIV infect dendritic cells, macrophages, and T cells in vivo.
Despite the apparent complexities of the chemokine signaling systems, the importance of individual chemokine receptors is gradually emerging from detailed studies on knockout mice, targeted gene disruption and the application of specific chemokine antagonists. As an example, CCR1 knockout mice have been reported to have disordered trafficking and proliferation of myeloid progenitor cells and to display impaired inflammatory responses to a variety of stimuli. Control of the CCR1 signaling system was demonstrated to have clinical significance as CCR1 knockout mice display significantly reduced rejection responses to cardiac allografts. This suggests that a strategy of blocking CCR1 signaling pathways may be useful in preventing rejection of transplanted tissues (ACTA BIOCHIMICA et BIOPHYSICA SINICA 2003, 35(9) :779-788).
CCR5 has generated widespread interest because of its role as a co-receptor for HIV. The identification of a naturally occurring mutant of this receptor, CCR5Δ32, and observations that homo and heterozygotes for this mutant have increased resistance to HIV infection and the development of AIDS has highlighted the potential benefits to human health that could accrue from controlling the ability of CCR5 to bind ligands (ACTA BIOCHIMICA et BIOPHYSICA SINICA 2003, 35(9):779-788).
Immunotherapy
Costimulatory molecules are important regulators of T cell activation and thus are the favored targets for therapeutic manipulation of the immune response. One of the key costimulatory receptors is CD80, which binds T cell ligands, CD28, and CTLA-4. It has been shown that expression of the costimulatory molecules CD80, CD86 and CD83 plays an important role in adjuvant activity and it is known that expression of CD86 is a feature of CT-based adjuvants (Lyke, 2004). Thus, molecules or compounds that affect CD80 expression represent promising novel therapeutic and immunotherapy agents that might induce protective immunity. A number of immunomodulatory therapies are being developed for clinical applications. These include approaches targeting antigen presentation and costimulation, T cell activation, action of proinflammatory mediators and modulating the cytokine balance (Asadullah et al., 2002). Tumor necrosis factors (TNFs) are known to be cytotoxic cytokines produced by macrophages and lymphocytes and are found to be suppressed in cancer patients or those who are pregnant.
Immunotherapy for Cancer
Immunosuppression is a hallmark of advanced malignancies in man (Lentz, 1999). Immunotherapy is the name given to cancer treatments that use the immune system to attack cancers. That is, the immune system can be stimulated to slow down the growth and spread of cancer. Immunotherapies involving certain cytokines and antibodies have now become part of standard cancer treatment. Immunotherapy of cancer began approximately 100 years ago when Dr. William Coley showed that cancer could be controlled by injections of bacterial products and components known as Coley's toxin. It is now known that the active anti-cancer component of Coley's toxin are bacterial oligonucleotides.
Systemic immunotherapy refers to immunotherapy that is used to treat the whole body and is more commonly used than local immunotherapy which is used to treat one “localized” part of the body, particularly when a cancer has spread. The suppressive milieu present within established tumors inhibits effective immune responses and new strategies are emerging to manipulate the local tumor environment to promote a proinflammatory environment, promote dendritic cell activation, and enhance antitumor immunity (Kaufman and Disis, 2004).
Immunotherapy is a potential useful strategy for the treatment of brain tumors because it offers a degree of specificity, the ability to extravasate into solid tumors, and the potential for eliciting a long-term protective immune response. Several approaches have been developed including the use of cytokines. In studies on the treatment of brain tumors, T cell stimulation with the proinflammatory cytokine IL-12 can elicit antitumor immunity (Gawlick et al., 2004). As such, cytokine treatments combined with tumor-targeted costimulation, or methods that stimulate cytokine production and the proinflammatory response, may be a useful adjunct treatment for brain tumors.
Immunotherapy for Infectious Diseases
In order to combat the increasing prevalence of drug-resistant Mycobacterium tuberculosis infection, new drugs are being developed. One promising strategy is to treat patients with refractory mycobacteriosis using ordinary antimycobacterial drugs in combination with appropriate immunomodulators in order to mobilize the cytokine network in response to mycobacterial infection such as using immunomodulating cytokines (especially Th-1 and Th-1-like cytokines such as IL-12 and proinflammatory cytokines such as TNF-α (Tomioka 2004). The Th-1 response participates in cell-mediated immunity and is essential in controlling infections due to intracellular pathogens and viruses.
Although Cryptococcus neoformans is a fungal pathogen that causes human disease predominantly in the immunocompromised host, severe infection can occur in immunocompetent individuals. Activation of cellular immunity plays a key role in anticryptococcal defense, and therefore, immunotherapy to increase the immune and proinflammatory response would be a useful treatment to restore immunological parameters and sustained clinical recovery for refractory cryptococcal meningitis (Netea et al., 2004).
The bacterium Bacillus anthracis causes the disease anthrax, which if left untreated, can result in bactermia, multisystem dysfunction and death. Anthrax lethal toxin severely impairs the function of dendritic cells—which are pivotal to the establishment of immunity against pathogens—and host immune responses (Agrawal et al., 2003). Dendritic cells exposed to lethal toxin and then exposed to lipopolysaccharide do not upregulate costimulatory molecules, secrete greatly diminished amounts of proinflammatory cytokines, and do not effectively stimulate T cells (Agrawal et al., 2003). Methods to stimulate dendritic cells and the proinflammatory response might be a useful strategy to stimulate the immune response and in the immunotherapy of anthrax infection.
Host defenses against systemic mycoses is multifactorial, depending on innate, as well as acquired mechanisms in which innate resistance includes inflammatory responses whereby production of proinflammatory cytokines increase the capacity of host defenses for killing (Clemons and Stevens, 2001). Therefore, a strong Th-1 response can provide protective immunity suggesting that immunotherapy has utility as a basis in treating or inhibiting mycoses.
Studies on the intracellular activities occurring during Salmonella infection in DCs show that the bacteria suppress T cell proliferation (Cheminay et al., 2005). This suggests that immunotherapy might be a useful approach in the inhibition or treatment of infections caused by intracellular bacteria such as Salmonella. 
Chemokines that bind to HIV co-receptors are potent and selective inhibitors of HIV infection and can be used in controlling HIV infection in concert with humoral and cellular immune and inflammatory responses (Garzino-Demo et al., 2000). This indicates that methods or molecules that promote the immunostimulation of chemokines can be used to inhibit or treat HIV infection.
Klebsiella pneumoniae is an encapsulated, highly virulent Gram-negative bacteria that is a leading cause of both community-acquired and nosocomial pneumonia. A frequent complication of pulmonary infection due to K. pneumoniae is the propensity of this organism to spread from the lung into the bloodstream, resulting in widespread systemic dissemination and death. Innate immunity is the principal pathway for elimination of virulent extracellular Gram-positive and Gram-negative pathogens, including K. pneumoniae, from the lung (Nelson et al., 1995). The two main phagocytic cells that constitute pulmonary innate immunity are resident alveolar macrophages (AM) and recruited neutrophils (PMN) (Lipscomb et al., 1983; and Towes et al., 1980). Both cell types are essential in host defense against bacterial pneumonia, such as that caused by K. pneumoniae, as the selective depletion of either cell population results in profound defects in the clearance of bacteria from the alveolar space (Broug-Holub et al., 1997; and Tsai et al., 2000). In addition, local and rapidly recruited lung DC internalize bacteria, which promotes DC maturation, expression of type 1 promoting cytokines (e.g., interleukin 12 (IL-12), type 1 interferons, and chemokines), co-stimulatory molecules, and migration to regional lymph nodes (Banchereau et al., 1998; Kikuchi et al., 2005; Kradin et al., 2000; Liu et al., 2006; and Mc William et al., 1994). Presentation of microbial antigens to naive T cells leads to the antigen-specific production of interferon-gamma (IFN-gamma) and the development of humoral immunity. Interferon-gamma can also be expressed early in infection in a non-antigen specific fashion by lung macrophages, NK cells, NKT cells, and γδ T cells, either directly in response to microbial signals or in a paracrine fashion in response to host-derived cytokines such as IL-12 (Deng et al., 2004; Ferlazzo et al., 2003; Johnston et al., 2003; and Moore et al., 2000). The type 1 cytokines IL-12, IFN-gamma, and IP-10 are required for host defense against both intracellular and extracellular bacterial pathogens (Brieland et al., 1998; Deng et al., 2001; Greenberger et al., 1996; Moore et al., 2002; Skerrett et al., 1994; Tateda et al., 1998 and 2001; Zeng et al., 2005; and Yoshida et al., 2001). Molecules that modulate (e.g., stimulate) the immune response might have clinical application in the inhibition (pretreatment) or therapy (treatment) of pneumonia.
Oligonucleotide Molecules as Anti-Cancer Agents
The use of unmethylated (CpG) oligonucleotides in the treatment or prevention of cancer has been reported. Synthetic oligonucleotides containing CpG with appropriate flanking regions (CpG motif) have been found to activate macrophages, dendritic cells and B cells to secrete a variety of immunomodulatory cytokines such as IL-6, IL-12, IL-18 and gamma interferon (Krieg, 2002). CpG DNA has also been shown to activate costimulatory molecules such as CD80 and CD86. CpG DNA induces strong innate immunity at mucosal surfaces. The immunostimulatory property of CpG DNA produces long-term vaccine-like effects due to its adjuvant properties. CpG oligonucleotides influence both antibody and cell-mediated immunity and applications include vaccine adjuvants, taming allergic reactions and potentiating monoclonal antibodies and cytotoxic immune cells. They also enhance the antitumor effects of chemotherapeutic agents and improve survival after surgical section of a solid tumor (Weigel et al., 2003). For CpG oligonucleotides, the anti-tumor effect is mediated via activation of the host immune system, not through direct anti-tumor effects. Data demonstrate that systemic application of proinflammatory reagents drastically enhances extravasation of effector cells into tumor tissue, an observation that is of general importance for immunotherapy of solid tumors in a clinical setting (Garbi et al., 2004). Based on their immunotherapeutic properties, CpG oligonucleotides have been used to treat and prevent various cancers and used in cancer vaccines. (U.S. Pat. Nos: 6,653,292; 6,429,199; 6,406,705; and 6,194,388).
Immunotherapy for Neurodegenerative Disease
The nervous system comprises the central and the peripheral nervous system. The central nervous system (CNS) is composed of the brain and spinal cord and the peripheral nervous system (PNS) consists of all of the other neural elements, namely the nerves and ganglia outside of the brain and spinal cord.
Damage to the nervous system may result from a traumatic injury, such as penetrating trauma or blunt trauma, or a disease or disorder, including but not limited to Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), diabetic neuropathy, senile dementia, and ischemia.
Maintenance of central nervous system integrity is a complex “balancing act” in which compromises are struck with the immune system. In most tissues, the immune system plays an essential part in protection, repair, and healing. In the central nervous system, because of its unique immune privilege, immunological reactions are relatively limited (Streilein, 1993 and 1995). A growing body of evidence indicates that the failure of the mammalian central nervous system to achieve functional recovery after injury is a reflection of an ineffective dialog between the damaged tissue and the immune system. For example, the restricted communication between the central nervous system and blood-borne macrophages affects the capacity of axotomized axons to regrow; transplants of activated macrophages can promote central nervous system regrowth (Lazarov Spiegler et al, 1996; Rapalino et al, 1998).
Activated T cells have been shown to enter the central nervous system parenchyma, irrespective of their antigen specificity, but only T cells capable of reacting with a central nervous system antigen seem to persist there (Hickey et al, 1991; Werkele, 1993; Kramer et al, 1995). T cells reactive to antigens of central nervous system white matter, such as myelin basic protein (MBP), can induce the paralytic disease experimental autoimmune encephalomyelitis (EAE) within several days of their inoculation into naive recipient rats (Ben-Nun, 1981a). Anti-MBP T cells may also be involved in the human disease multiple sclerosis (Ota, K. et al, 1990; Martin, 1997). However, despite their pathogenic potential, anti-MBP T cell clones are present in the immune systems of healthy subjects (Burns, 1983; Pette, M. et al, 1990; Martin et al, 1990; Schluesener et al, 1985). Activated T cells, which normally patrol the intact central nervous system, transiently accumulate at sites of central nervous system white matter lesions (Hirschberg et al, 1998).
A catastrophic consequence of central nervous system injury is that the primary damage is often compounded by the gradual secondary loss of adjacent neurons that apparently were undamaged, or only marginally damaged, by the initial injury (Faden et al, 1992; Faden 1993; McIntosh, 1993). The primary lesion causes changes in extracellular ion concentrations, elevation of amounts of free radicals, release of neurotransmitters, depletion of growth factors, and local inflammation. These changes trigger a cascade of destructive events in the adjacent neurons that initially escaped the primary injury (Lynch et al, 1994; Bazan et al, 1995; Wu et al, 1994). This secondary damage is mediated by activation of voltage-dependent or agonist-gated channels, ion leaks, activation of calcium-dependent enzymes such as proteases, lipases and nucleases, mitochondrial dysfunction and energy depletion, culminating in neuronal cell death (Yoshina et al, 1991; Hovda et al, 1991; Zivin et al, 1991; Yoles et al, 1992). The widespread loss of neurons beyond the loss caused directly by the primary injury has been called “secondary degeneration.”
One of the most common mediators which cause self-propagation of the diseases even when the primary risk factor is removed or attenuated is glutamate, an excitatory amino acid capable of displaying dual activity: playing a pivotal role in normal central nervous system (CNS) functioning as an essential neurotransmitter, but becoming toxic when its physiological levels are exceeded. Elevation of glutamate has been reported in many CNS disorders. In its role as an excitotoxic compound, glutamate is one of the most common mediators of toxicity in acute and chronic (including optic nerve degeneration in glaucoma) degenerative disorders (Pitt et al., 2000 and Schoepp et al., 1996). Endogenous glutamate has been attributed to the brain damage occurring acutely after status epilepticus, cerebral ischemia or traumatic brain injury. It may also contribute to chronic neurodegeneration in such disorders as amyotrophic lateral sclerosis and Huntington's chorea.
Intensive research has been devoted to attenuating the cytotoxic effect of glutamate by the use of locally acting drugs, such as NMDA-receptor antagonists (Brauner-Osborne et al., 2000). Conventional therapy of this type is often unsatisfactory, however, as in neutralizing the toxic effect it is likely to interfere with the physiological functioning. In humans, such compounds have psychotropic and other side effects that make them unsuitable as therapeutic agents. They also have the disadvantage of interfering with the essential physiological functioning of glutamate as a ubiquitous CNS neurotransmitter. Because glutamate activity is essential for normal physiological functioning, yet is potentially devastating after acute injury or in chronic CNS disorders, any attempt to neutralize its harmful effect must do so without eliminating its essential activity at other sites in the body.
Another tragic consequence of central nervous system injury is that neurons in the mammalian central nervous system do not undergo spontaneous regeneration following an injury. Thus, a central nervous system injury causes permanent impairment of motor and sensory functions.
Spinal cord lesions, regardless of the severity of the injury, initially result in a complete functional paralysis known as spinal shock. Some spontaneous recovery from spinal shock may be observed, starting a few days after the injury and tapering off within three to four weeks. The less severe the insult, the better the functional outcome. The extent of recovery is a function of the amount of undamaged tissue minus the loss due to secondary degeneration. Recovery from injury would be improved by neuroprotective treatment that could reduce secondary degeneration. For example, alleviation of the effect of glutamate is a frequent target of neuroprotective drug development. Among the drugs which are being developed for this purpose are N-methyl-D-aspartate (NMDA)-receptor or alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA)-receptor antagonists. These drugs will inevitably have severe side effects as they interfere with the functioning of NMDA and AMPA receptors, which are crucial for CNS activity. One of the most intensely studied NMDA-receptor antagonists is MK801, which provides effective neuroprotection but with severe side effects. In animal models of cerebral ischemia and traumatic brain injury, NMDA and AMPA receptor antagonists protect against acute brain damage and delayed behavioral deficits. Such compounds are undergoing testing in humans, but therapeutic efficacy has yet to be established. Other clinical conditions that may respond to drugs acting on glutamatergic transmission include epilepsy, amnesia, anxiety, hyperalgesia and psychosis (Meldrum, 2000).
Glaucoma may be viewed as a neurodegenerative disease and consequently amenable to any therapeutic intervention applicable to neurodegenerative diseases. There is evidence that neuroprotection can be achieved both pharmacologically and immunologically where immunologic intervention boosts the body's repair mechanisms for counteracting the toxicity of physiologic compounds acting as stress signals and that boosting of a T cell-based mechanism promotes recovery of the damaged optic nerve. (Schwartz, 2003; Schwartz, 2004).
In rat cerebral cortical cultures, neuronal killing was partially or completely prevented by chemokines that stimulate the CXCR4, CCR3 or CCR5 chemokine receptors (Brenneman et al., 1999). Cytokines have been shown to be involved in nerve regeneration (Stoll et al., 2000).
Vaccines and Adjuvants
Vaccination is the single most valuable tool in the prevention of disease caused by infectious agents. Vaccination to protect against various infectious diseases may be enhanced by using adjuvants that can selectively stimulate immunoregulatory responses. Compared to injection of an antigen alone, injection of antigen plus an adjuvant generally permits use of a much smaller quantity of antigen and increases the antibody titer. Attenuated viruses and recombinant proteins are poorly immunogenic and absolutely require adjuvants for efficient immunostimulation, as do other antigens such as synthetic peptides, subunit vaccines, polysaccharides, killed cell preparations and plasmid DNA. For example, tetanus toxoid is not immunogenic in the absence of adjuvants. Some of these antigens require high production costs due to purification processes that are necessary to avoid contamination from cell products. The adjuvant may aid the immune response by forming a depot of antigen at the site of interest, it may serve as a vehicle to help deliver the antigen to the spleen or lymph nodes where antigen is trapped by follicular DCs, or it may activate the various cells involved in the immune response, either directly or indirectly. Many bacteria contain substances or products (e.g., endotoxin or cell wall constituents) that activate cells of the immune system. Safe and potent new adjuvants are needed for vaccines. These include vaccines that are administered at mucosal surfaces. The development of methods to enhance antigen presentation by DC is required for successful vaccines, particularly in immunocompromised patients. Activation of DCs is crucial for priming cytotoxic T lymphocytes (CTL), which have a critical role in tumor immunity, and it is considered that adjuvants are necessary for activation of DCs and for enhancement of cellular immunity. A Th-1 oriented immune response is important for an adequate cell mediated immune response and for protection induced by natural infection or vaccination with vaccines. Desirable properties of an adjuvant other than a strong and sustained immunostimulatory ability that should be considered are its safety, biodegradability, stability, ease of mixing and use, broad range of antigens and administration routes that can be used, and its economical manufacture.
A number of adjuvants have been developed. Complete Freund's adjuvant (FCA) is a mixture of a non-metabolizable oil (mineral oil), a surfactant, and killed mycobacterial cells and has been used for many years to enhance the immunologic responses to antigens. Although FCA is effective for production of antibodies, there are problems and hazards associated with its use including a chronic inflammatory response at the site of injection that may be severe and painful which might result in granulomas (Broderson, 1989). FCA is also a hazard for laboratory personnel (Chapel and August, 1976). Incomplete Freund's adjuvant (FIA) does not contain any mycobacterial cells and while it shows adjuvant properties, it is considered less potent than FCA. A number of experimental adjuvants have been reported in recent years (McCluskie and Weeratna, 2001) which include: bacterial toxins such as cholera toxin (CT), Escherichia coli labile toxin (LT), IL-12, LPS-derivatives, and oligonucleotides containing CpG motifs. Their mode of action differ but include: a) enhancement of immunological half-life of the co-administered vaccine antigen; b) increased antigen uptake and presentation; and c) modulatory effects on the production of immunomodulatory cytokines resulting in the preferential development of certain types of immune responses (e.g., Th-1 versus Th-2, mucosal, cell mediated, etc). Adjuvants can be classified into two groups: i) immunostimulatory molecules such as CpG oligonucleotides, bacterial toxins and derivatives, the lipopolysaccharide derivative lipid A, cytokines and hormones; and ii) delivery systems which possess inherent immunostimulatory activity such as liposomes, emulsions, microparticles.
With cancer vaccines, the objective is to get the body to elicit its own immune response. Cancer vaccines would typically consist of a source of cancer-associated material or cells (antigen) that may be autologous (from self) or allogenic (from others) to the patient, along with other components (e.g., adjuvants) to further stimulate and boost the immune response against the antigen. Cancer vaccines cause the immune system to produce antibodies to one or several specific antigens, and/or to produce killer T cells to attack cancer cells that have those antigens. T cells in the body react with cancer cells so stimulation of a patient's T cells would increase the ability of T cells to recognize cancer cells. In addition, dendritic cells which are specialized antigen presenting cells, help the immune system to recognize cancer cells by presenting cancer antigens to T cells, making it easier for the immune system cells to react with and attack them. Dendritic cells are the most effective antigen-presenting cells known. Dendritic cells link innate immunity and adaptive immunity. Dendritic cells can efficiently present cancer proteins to activate the immune response, so agents that activate or turn on dendritic cells and the immune response, have clinical applications in preventing or treating cancer and in immunotherapy.
Studies on antitumor immunity have shown that a nontoxic cholera toxin subunit can up-regulate the secretion of IL-12 from DCs suggesting DC maturation and that this molecule acts as an adjuvant to enhance immunity through DC maturation and may be considered a useful adjuvant to raise immunity in a clinical application (Isomura et al., 2005). IL-12 can act as a mucosal adjuvant for coadministered antigens. Studies have shown that proinflammatory cytokines such as IL-12 can replace cholera toxin (CT) as a mucosal adjuvant for antibody induction and are important candidates for use as mucosal adjuvants with HIV and other vaccines (Bradney et al., 2002).
DNA containing an unmethylated CpG motif (CpG oligonucleotides) are a potent immunostimulator and can trigger innate immune responses which promote the combating of infection. Oligonucleotides containing unmethylated CpG motifs act as immune adjuvants, accelerating and boosting antibody responses promoting the production of Th-1 proinflammatory cytokines and inducing the maturation/activation of DCs (Klinman, 2003). CpG oligonucleotides have become a promising immunotherapeutic candidate to assist and direct immune responses such as vaccination or modulation of allergic responses (Dalpke, et al., 2002). CpG oligonucleotides are a strong inducer of IL-12 indicating that it acts as a Th-1 polarizing agent that can be utilized as a potent vaccine adjuvant (Dalpke et al., 2002). Infection such as those caused by intracellular bacteria and viruses, induces innate immunity by causing the infected cells to produce proinflammatory cytokines that directly combat bacterial invaders and to express costimulating surface molecules, which develop adaptive immunity by inducing T cell differentiation. CpG DNA immunostimulatory responses are consistent between in vitro and in vivo studies (Zelenay et al., 2003). Coadministration of CpG DNA with a variety of vaccines has improved protective immunity in animal challenge models and are safe and well-tolerated (Klinman, 2003). A study addressing tumor immune therapy has shown that stimulation of T helper cells with syngeneic tumor cells and antigen-presenting cells in the presence of CpG DNA allows the generation of large numbers of strongly polarized, tumor-specific Th-1 cells, indicating the eradication of established tumors and lymphoma by activating proinflammatory responses and based on this immunostimulatory ability, has clinical utility in immunotherapy (Egeter et al., 2000).
While certain treatments for infectious diseases, cancer, immunodeficiciency and inflammatory disorders and neurological and neurodegenerative diseases are available, improved treatments are needed. Also needed are the development of improved vaccines for a variety of diseases through the use of better vaccine adjuvants.
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